Hi,
Currently I'm doing TFC assay with quercetin for a standard curve. 1 ml of quercetin solution (10, 20, 40, 60, 80, 100, 120 ppm) was added with 4 ml of distilled deionized water (dd H2O) and 0.3 ml of 5% NaNO3. After 5 minutes, 0.3 ml of 10% of AlCl3 was added and at the sixth minute, 2 ml of 1M NaOH was added and total volume was made up to 10 ml with dd H2O.
Quercetin was diluted with 100% methanol. The rest was diluted with dd H2O.
Blank: 1 ml methanol + 4ml ddH2O + 0.3 ml of 5% NaNO3 + 0.3 ml of 10% of AlCl3 + 2 ml of 1M NaOH + dd H2O
I obtained negative reading of absorbance with the R2 was 0.92.
10 ppm = -0.1248
20 ppm = -0.1663
40 ppm = -0.3286
60 ppm = -0.5434
80 ppm = -0.5132
100 ppm = -0.9331
120 ppm = -0.8836
Are the above procedure is correct?
Ref: Atanassova M., Georgieva S., Ivancheva K., Total Phenolic and total flavonoid contents, antioxidant capacity and biological contaminants in medicinal herbs., Journal of the University of Chemical Technology and Metallurgy, 46,1,2011, 81-88.
supposed to get positive abs value. refer the attached paper. they used the same method as yours and obtained positive abs values... what was the color of final solution? is the blank clear?
Dear Haiza Muda,
You are suppose to obtain positive result, it means your result is negative correlation with concentration. However, try higher concentration like 1, 2, 4, 8, 10, 20 ug/ml. I feel convenient in micro-gram than ppm.
Negative value of absorbance implies that the blank reading is more than that of the sample.
You are supposed to have a deeper color development for the sample so that when you subtract the absorbance of the reagents you will be left with the exact absorbance of the sample.
Please repeat the procedure carefully while ensuring you prepare the blank same time as the sample.
I used the same protocol and I obtained positive values. I suggest for you the use of higher concentrations (100- 120-140-160-180-200 ppm).
Try higher concentrations of quercetin in mg/ml not in ppm. Use a quercetin that is of analytical grade, i am suggesting that you buy a Sigma product
Thank you all for the answers. I already repeat the same protocol and managed to obtained the positive abs.
Hi,
You should prepare the blank without AlCl3, not without quercetin, read the method with attention. Or see another articles.
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